TWI479153B - Screening kit for screening anti - influenza drugs and its screening method - Google Patents
Screening kit for screening anti - influenza drugs and its screening method Download PDFInfo
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本發明係有關於藥物篩檢方法，特別係指一種抗流感候選藥物之篩選方法及其套組。The invention relates to a drug screening method, in particular to a screening method for anti-influenza drug candidates and a kit thereof.
按，流行性感冒係一種存在於家禽或是哺乳動物間之傳染性疾病，由屬於正黏液病毒科(Orthomyxoviridae )之流行性感冒病毒所引起。一般來說，流行性感冒典型會引起急性上呼吸道感染，並伴隨著上下呼吸道發炎現象，而臨床症狀包含有發燒、喉嚨痛、肌肉酸痛、頭痛、咳嗽、虛弱等，會使病患產生不舒服以及虛弱之感受。於較為嚴重之情形下，流行性感冒係會引起肺炎，而使全球每年有超過30萬之小孩以及老年人死亡。更進一步而言，人類流感病毒可分為三類，分別為A型流感病毒、B型流感病毒以及C型流感病毒，其中，以A型流感病毒具有極強變異性，對於大眾健康具最嚴重之威脅性，常見之A型流感病毒為西班牙流感(H1N1)、亞洲流感(H2N2)以及香港流感(H3N2)，而西班牙流感係為人類史上最致命之流行性感冒。最近一次大流行係於2009年，豬源性A型流感病毒或稱新型流感病毒(swine origin influenza virus；SOIV)普遍地被散佈於世界各地，並引起1萬8千人死亡。因此，為了對抗流行性感冒，科學家無一不致力於相關治療方法之研究。According to the epidemic, an infectious disease that exists between poultry or mammals is caused by an influenza virus belonging to the Orthomyxoviridae family. In general, influenza usually causes acute upper respiratory tract infections, accompanied by inflammation of the upper and lower respiratory tract, and clinical symptoms include fever, sore throat, muscle aches, headache, cough, weakness, etc., which can cause discomfort to the patient. And the feeling of weakness. In more serious cases, influenza can cause pneumonia, which kills more than 300,000 children and the elderly every year. Furthermore, human influenza viruses can be divided into three categories, namely influenza A virus, influenza B virus and influenza C virus. Among them, influenza A virus has strong variability and is most serious for public health. Threatened, the common influenza A viruses are Spanish flu (H1N1), Asian flu (H2N2) and Hong Kong flu (H3N2), while the Spanish flu is the most deadly influenza in human history. The most recent pandemic was in 2009. The swine-type influenza A virus or the new swine origin influenza virus (SOIV) was widely spread around the world and caused 18,000 deaths. Therefore, in order to fight against influenza, scientists are committed to the study of related treatment methods.
流行性感冒病毒之結構自內到外主要可分為核心、基質蛋白以及包膜三部份，其中，核心係包含儲存病毒訊息之遺傳物質與複製該訊息所需之酵素；基質蛋白係構成病毒之外殼骨架，用以緊密結合最外層之包膜，保護病毒核心以及維持病毒空間結構；包膜則是包裹於基質蛋白外之一層磷脂層膜，而於病毒外膜與磷脂雙層膜間係具有紅血球凝聚素(hemagglutinin；HA；H)以及神經氨酸酶(neuraminidase；NA；N)，分別扮演侵入宿主細胞以及與宿主細胞分離，於病毒感染宿主之過程中 扮演著重要角色。故，先前研究係以可被人類血清中的抗體辨識之該二醣蛋白作為抗病毒藥物之主要目標。目前最為人所熟知之抗流感藥物克流感(Tamiflu)，係為一種神經氨酸酶抑制劑，惟，由於新型流感病毒所產生神經氨酸酶之突變(H274Y)，對於克流感該種藥物具有抗性。是以，目前科學家急需研究一種新且有效之抗流感藥物。The structure of influenza virus can be divided into three parts: core, matrix protein and envelope. The core contains the genetic material for storing viral information and the enzyme needed to replicate the message. The matrix protein constitutes a virus. The shell skeleton is used to tightly bond the outermost envelope to protect the virus core and maintain the spatial structure of the virus; the envelope is encapsulated in a layer of phospholipid membrane outside the matrix protein, and between the outer membrane of the virus and the phospholipid bilayer membrane Hemagglutinin (HA; H) and neuraminidase (NA; N), which act as invasive host cells and separate from host cells, in the process of virus infection of the host Play an important role. Therefore, previous studies have identified this disaccharide protein as an antiviral drug with antibodies recognized by human serum. The most well-known anti-influenza drug, Tamiflu, is a neuraminidase inhibitor. However, due to the mutation of neuraminidase produced by the novel influenza virus (H274Y), this drug has Resistance. Therefore, scientists are urgently needed to study a new and effective anti-influenza drug.
先前研究指出A型流感病毒結構蛋白中之核殼蛋白(Nucleocapsid，N)含有498個胺基酸，分子量約為56kDa，其係與RNA在病毒粒子中結合呈螺旋狀，參與病毒感染、複製和病毒包裝。核殼蛋白係為一個三聚體之蛋白質，主要之功用在於和病毒RNA基因組結合，形成20個核苷酸之核醣蛋白複合體(ribonucleoprotein complexes；RNP)，並在生物接觸過程中扮演病毒與宿主細胞之間關鍵之接合器。核殼蛋白之單體構型似月牙形，具有四個區域，其中，其頭部(head domain)與身部(body domain)間具有凹槽，係為與RNA相結合之區域。而核殼蛋白除了與RNA作用外，亦與細胞多胜肽產生交互作用，包括肌動蛋白、進核與出核訊息以及細胞內之RNA解旋酶(nuclear RNA helicase)。如上所述，A型流感病毒之核殼蛋白含有498個胺基酸，而該498個胺基酸也高度相似地存在於流感病毒中，並於病毒生長週期中具有多功性，使核殼蛋白成為抗流感藥物發展之新對象。Previous studies have indicated that the nucleocapsid protein (Nucleocapsid, N) in the structural protein of influenza A virus contains 498 amino acids with a molecular weight of approximately 56 kDa, which binds to RNA in the virion and is involved in viral infection, replication and Virus packaging. The nucleocapsid protein is a trimeric protein that functions primarily with the viral RNA genome to form a 20-nucleotide ribonucleoprotein complexes (RNP) that acts as a virus and host during biological contact. A key adapter between cells. The nucleocapsid protein has a monomeric configuration like a crescent shape and has four regions in which a recess between a head domain and a body domain is a region that binds to RNA. In addition to its interaction with RNA, nucleocapsid protein also interacts with cellular peptides, including actin, nuclear and nuclear information, and intracellular RNA helicase. As mentioned above, the nucleocapsid protein of influenza A virus contains 498 amino acids, and the 498 amino acids are also highly similar in influenza virus and have pluripotency in the virus growth cycle, making the core shell Protein has become a new target for the development of anti-influenza drugs.
然而，開發新藥必須要投入大量金錢以及時間，根據美國所作統計，開發新藥上市前平均需要投入5億美元之資金，耗時約12~15年，而研製新藥中僅有不到百分之五能夠進入臨床前研究階段，其中僅有百分之二能夠進入臨床試驗階段，其中又有百分之八十會在上市前被淘汰，因此，如何事先對於欲開發之新藥物有所評估，有效率篩選出可能具有療效新型藥物，再做進一步開發研究，亦即縮短開發新型藥物週期，提高藥物開發效率以及投入資金之回收率。目前已有研發出縮短藥物開發週期之技術，例如建立數據資料庫，藉由比對、分析以及評定， 於進行實驗前先應用程式篩選掉不太可能成功之藥物，亦可用以預測候選藥物於體內吸收、代謝、毒性等，降低研發新藥之淘汰率。惟，利用資料庫之方式必須先瞭解候選藥物之分子結構與特點，以及需預先建立完整資料庫，才能增加比對分析之準確率，如此一來，對於研究單位或是業者而言，執行操作上仍有一定難度。因此，開發一種簡易操作又有效率之藥物篩選方法，其係為絕大多數藥廠最急切需要之研究目標。However, the development of new drugs requires a lot of money and time. According to statistics from the United States, it takes an average of 500 million U.S. dollars to develop new drugs before they go on the market. It takes about 12 to 15 years, and less than 5 percent of new drugs are developed. Being able to enter the preclinical research phase, only 2% of them can enter the clinical trial stage, and 80% of them will be eliminated before going public. Therefore, how to evaluate the new drugs to be developed in advance, there are Efficacy screens for new drugs that may be effective, and further research and development, that is, shortening the development of new drug cycles, improving drug development efficiency and recovery of investment funds. Techniques for shortening the drug development cycle have been developed, such as the establishment of data repositories, through comparison, analysis and assessment, Before the experiment, the application screens out the drugs that are unlikely to be successful, and can also be used to predict the absorption, metabolism, toxicity, etc. of the candidate drugs in the body, and reduce the elimination rate of new drugs. However, the use of the database must first understand the molecular structure and characteristics of the candidate drug, and the need to establish a complete database in advance, in order to increase the accuracy of the comparison analysis, so that for the research unit or the industry, the operation There is still some difficulty in it. Therefore, the development of a simple and efficient drug screening method is the most urgent research goal of most pharmaceutical companies.
因此，本發明之主要目的即在於提供一種抗流感候選藥物之篩選套組，其係包含有一載具，一預定濃度之核殼蛋白溶液，添加於該載具中，據此，將一待測藥物添加入該載具中，與該核殼蛋白溶液相結合，使之表現不同螢光強度，用以篩選該待測藥物，其中：該核殼蛋白溶液係包含有一經純化後之重組核殼蛋白；該載具係可設為一多孔盤，而該核殼蛋白溶液依據需求添加一定量於該多孔盤中之各孔中。本發明之另一目的係在於提供一種抗流感候選藥物之篩選方法，包含下列步驟：Therefore, the main object of the present invention is to provide a screening kit for an anti-influenza drug candidate, which comprises a carrier, a predetermined concentration of a nucleocapsid protein solution, added to the carrier, according to which a test is to be performed The drug is added to the carrier and combined with the nucleocapsid protein solution to exhibit different fluorescence intensity for screening the drug to be tested, wherein: the nucleocapsid protein solution comprises a purified recombinant core shell The protein; the carrier can be configured as a porous disk, and the nucleocapsid protein solution is added in an amount to each well in the porous disk as needed. Another object of the present invention is to provide a screening method for an anti-influenza drug candidate, comprising the following steps:
步驟a：製備一預定濃度之核殼蛋白溶液。Step a: preparing a predetermined concentration of nucleocapsid protein solution.
步驟b：取一待測藥物，添加於該核殼蛋白溶液中。Step b: taking a drug to be tested and adding it to the nucleocapsid protein solution.
步驟c：檢測該核殼蛋白溶液螢光強度之變化，用以篩選出具有抗流感能力之藥物。Step c: detecting changes in the fluorescence intensity of the nucleocapsid protein solution for screening for drugs having anti-influenza ability.
更進一步而言，該步驟a更包含有：Further, the step a further includes:
步驟a1：構築一重組核殼蛋白之表現載體，包含有一編碼為核殼蛋白之核苷酸序列，其中，該編碼為核殼蛋白之核苷酸序列係以預定限 制酶截切選自於命名為H1N1A/TW/22/2001之台灣種新型流感病毒之核殼蛋白或是豬源性A型流感病毒之核殼蛋白(swine-origin influenza A)而得。Step a1: constructing a expression vector of recombinant nucleocapsid protein, comprising a nucleotide sequence encoding a nucleocapsid protein, wherein the nucleotide sequence encoding the nucleocapsid protein is predetermined The enzyme cleavage is selected from the nucleocapsid protein of the Taiwanese novel influenza virus designated H1N1A/TW/22/2001 or the swine-origin influenza A of the porcine influenza A virus.
步驟a2：將該表現載體轉型至一宿主內，使該載體表現該重組核殼蛋白，其中，宿主可為一細菌。Step a2: The expression vector is transformed into a host such that the vector exhibits the recombinant nucleocapsid protein, wherein the host can be a bacterium.
步驟a3：萃取並純化該重組核殼蛋白，將之製備成為一預定濃度之重組核殼蛋白溶液。Step a3: The recombinant nucleocapsid protein is extracted and purified, and prepared into a predetermined concentration of recombinant nucleocapsid protein solution.
藉由本發明所提供抗流感候選藥物之篩選方法，可簡化篩選待測藥物之過程，縮短研發新型藥物之週期，降低投入之研發資金，並提高新藥開發之成功率。By the screening method of the anti-influenza drug candidate provided by the invention, the process of screening the drug to be tested can be simplified, the cycle of developing new drugs can be shortened, the research and development funds can be reduced, and the success rate of new drug development can be improved.
本發明所揭一種抗流感候選藥物之篩選套組，其係包含有一載具，一預定濃度之重組核殼蛋白溶液於該載具內，得用以與一待測藥物相結合而表現出不同螢光強度，篩選出具有抗流感功能之候選藥物。此外，本發明更進一步提供抗流感候選藥物之篩選方法，首先，製備一預定濃度之核殼蛋白溶液，並取一待測藥物，添加於該核殼蛋白溶液中，使兩者混合形成一混合物，而後檢測該混合物之螢光強度，即可藉由分析螢光強度之變化而篩選出具有抗流感能力之候選藥物。The invention relates to a screening kit for anti-influenza drug candidates, which comprises a carrier, a predetermined concentration of recombinant nucleocapsid protein solution in the carrier, which can be used to combine with a drug to be tested to exhibit different Fluorescence intensity, screening for drug candidates with anti-influenza function. In addition, the present invention further provides a screening method for an anti-influenza drug candidate. First, a predetermined concentration of a nucleocapsid protein solution is prepared, and a drug to be tested is added to the nucleocapsid protein solution to mix the two to form a mixture. Then, by detecting the fluorescence intensity of the mixture, a candidate drug having anti-influenza ability can be selected by analyzing the change in fluorescence intensity.
需更進一步加以說明者，由新型流感病毒之核殼蛋白結構可知，於遠離其RNA結合處係具有六個色胺酸之殘基。而色胺酸本身係為重要之內在螢光探針，用以於微環境變化下偵測色胺酸變化而得知蛋白質之改變。簡言之，藉由核殼蛋白中色胺酸螢光強度之變化，可知核殼蛋白 是否與待測藥物相結合。Further to be explained, the nucleocapsid protein structure of the novel influenza virus is known to have residues of six tryptophan acids away from the RNA binding site. Tryptophan is itself an important intrinsic fluorescent probe for detecting changes in tryptophan under microenvironmental changes to detect protein changes. In short, nucleocapsid protein is known by the change in the fluorescence intensity of tryptophan in nucleocapsid protein. Whether it is combined with the drug to be tested.
以下，為了更進一步說明本發明，茲舉若干實例並配合圖式做更詳細說明如後。In the following, in order to further illustrate the present invention, a few examples will be described in more detail with reference to the drawings.
實例一：建構表現質體Example 1: Constructing a plastid
本實例係取由三軍總醫院以及長庚大學提供所提供之台灣種H1N1新型流感病毒之核殼蛋白以及豬源性A型流感病毒之核殼蛋白作為板模。This example is based on the nucleocapsid protein of the Taiwanese H1N1 new influenza virus and the nucleocapsid protein of the porcine avian influenza A virus provided by the Three Military General Hospital and Chang Gung University.
為了能夠獲得全長重組核殼蛋白，將該二核殼蛋白分別利用引子以聚合酶鏈反應(PCR)擴增。將經聚合酶鏈反應之各該產物以酵素截切於NdeI及XhoI兩切位，得到一核殼蛋白片段，並於各該核殼蛋白之N端連接六個組胺酸作為標記，而以T4連接酶構築於質體(pET21b)上，如第一圖所示。In order to obtain a full-length recombinant nucleocapsid protein, the dinucleocapsid protein was amplified by polymerase chain reaction (PCR) using primers, respectively. Each of the products of the polymerase chain reaction is cleaved by enzymes at both NdeI and XhoI sites to obtain a nucleocapsid protein fragment, and six histidine acids are linked to the N-terminus of each nucleocapsid protein as a marker. The T4 ligase is constructed on the plastid (pET21b) as shown in the first figure.
實例二：純化重組H1N1核殼蛋白以及重組SOIV核殼蛋白Example 2: Purification of recombinant H1N1 nucleocapsid protein and recombinant SOIV nucleocapsid protein
將實例一中所構築好之表現質體轉形至一含有乳糖操縱組之宿主細菌中，於1mM之異丙基半乳糖苷(IPTG)培養基、10℃培養24小時以誘導核殼蛋白產生。將完成培養之細菌以6000xg、4℃離心15分鐘，再以裂解緩衝液裂解細菌小球。而後，以13000xg、4℃離心30分鐘，移除沈澱物，而得到含有可溶性蛋白質之上清液。藉由液相層析法，將該上清液以鎳管柱、15~300mM咪唑洗提，而純化出N端帶有組胺酸標記之重組核殼蛋白，再以無咪唑之緩衝液透析經純化之片段並收集之。The plastids constructed in Example 1 were transformed into a host bacteria containing a lactose-administered group, and cultured in 1 mM isopropylgalactoside (IPTG) medium at 10 ° C for 24 hours to induce nucleocapsid production. The cultured bacteria were centrifuged at 6000 x g for 15 minutes at 4 ° C, and the bacterial pellet was lysed with a lysis buffer. Thereafter, the mixture was centrifuged at 13,000 x g for 30 minutes at 4 ° C, and the precipitate was removed to obtain a supernatant containing soluble protein. The supernatant was eluted with a column of nickel, 15 to 300 mM imidazole by liquid chromatography, and the recombinant nucleocapsid protein with a histidine-labeled N-terminal was purified and dialyzed against a buffer without imidazole. The purified fragments were collected and collected.
將純化後之台灣種H1N1新型流感病毒之重組核殼蛋白以及豬源性A型流感病毒之重組核殼蛋白分別以十二烷基硫酸鈉聚丙烯醯胺膠體電泳，利用考馬斯亮藍(Coomassie blue)之蛋白質定量分析法，結果如第二圖所示，其中，右邊為台灣種H1N1新型流感病毒之重組 核殼蛋白，左邊為豬源性A型流感病毒之重組核殼蛋白。Recombinant nucleocapsid protein of purified H1N1 influenza virus from Taiwan and recombinant nucleocapsid protein of porcine influenza A virus were electrophoresed with sodium dodecyl sulfate polyacrylamide colloid, respectively, using Coomassie blue The protein quantitative analysis method, the results are shown in the second figure, in which the right side is the reorganization of the new H1N1 influenza virus in Taiwan. The nucleocapsid protein, on the left, is the recombinant nucleocapsid protein of the swine-derived influenza A virus.
由第二圖之結果分析可得知該二重組核殼但白經純化後，純度高達90%以上，因此，將該二重組核殼蛋白供以下實例利用。It can be seen from the analysis of the results of the second figure that the purity of the two recombinant nucleocapsids but after purification of the white is up to 90% or more. Therefore, the two recombinant nucleocapsid proteins are utilized in the following examples.
實例三：以重組核殼蛋白檢測藥物Nucleozin(一)Example 3: Detection of Nucleozin by recombinant nucleocapsid protein (1)
需先加以說明者，於先前研究中已指出藥物Nucleozin具有連結新型流感病毒之核殼蛋白之能力，而使新型流感病毒喪失複製功能，亦即Nucleozin係為一種目前已知之抗流感藥物。因此，於本實例係利用藥物Nucleozin結合核殼蛋白，以觀察色胺酸所引起之螢光猝滅反應。To be explained first, it has been pointed out in previous studies that the drug Nucleozin has the ability to bind to the nucleocapsid protein of the novel influenza virus, and the novel influenza virus loses its replication function, that is, the Nucleozin system is a currently known anti-influenza drug. Therefore, in this example, the drug Nucleozin was used to bind to the nucleocapsid protein to observe the fluorescence quenching reaction caused by tryptophan.
首先，取該實例二中之台灣種H1N1新型流感病毒之重組核殼蛋白，製備成一最終濃度為4μM之重組核殼蛋白溶液。取濃度為2μM、4μM、20μM、40μM之藥物Nucleozin，分別添加於該重組核殼蛋白溶液中，形成待測樣品，其中，該核殼蛋白溶液之酸鹼值係為7.5，包含有50mM三羥基甲烷-氯化氫以及150mM氯化鈉。First, the recombinant nucleocapsid protein of the novel H1N1 influenza virus from Taiwan in Example 2 was prepared to prepare a recombinant nucleocapsid protein solution with a final concentration of 4 μM. The drug Nucleozin at a concentration of 2 μM, 4 μM, 20 μM, and 40 μM was separately added to the recombinant nucleocapsid protein solution to form a sample to be tested, wherein the nucleocapsid protein solution had a pH of 7.5 and contained 50 mM of trihydroxyl Methane-hydrogen chloride and 150 mM sodium chloride.
分別將不同濃度之待測樣品於25℃置放3小時後，以螢光光譜儀於波長300~400nm偵測各該待測樣品之螢光強度，結果如第三圖所示，其中，以未添加任何藥物之樣品作為對照組。The samples of different concentrations are placed at 25 ° C for 3 hours, and the fluorescence intensity of each sample to be tested is detected by a fluorescence spectrometer at a wavelength of 300 to 400 nm. The result is shown in the third figure, wherein A sample of any drug was added as a control group.
由第三圖之結果顯示，結果顯示於波長331nm時，未添加核殼蛋白之樣品具有220AU之螢光強度，而隨著添加藥物Nucleozin濃度之增加，於各該樣品中所測得之螢光強度隨之減弱。因此，可得知核殼蛋白與藥物Nucleozin間有連結反應，並隨著藥物Nucleozin濃度增加，與核殼蛋白間之連結亦隨之增加，造成核殼蛋白所產生之螢光強度降低，簡言之，螢光強度與藥物濃度間成反比的趨勢。As shown by the results of the third graph, the results show that at a wavelength of 331 nm, the sample without nucleocapsid protein has a fluorescence intensity of 220 AU, and the fluorescence measured in each sample increases with the increase of the concentration of the added drug Nucleozin. The intensity is reduced. Therefore, it can be known that there is a linkage reaction between the nucleocapsid protein and the drug Nucleozin, and as the concentration of the drug Nucleozin increases, the linkage with the nucleocapsid protein also increases, resulting in a decrease in the fluorescence intensity produced by the nucleocapsid protein. There is a tendency for the fluorescence intensity to be inversely proportional to the drug concentration.
實例四：以重組核殼蛋白檢測藥物Nucleozin(二)Example 4: Detection of Nucleozin with Recombinant Nucleocapsid Protein (II)
如同實例三所述流程，將濃度為4μM之藥物Nucleozin添加於重組核殼蛋白溶液中，形成待測樣品，並於25℃下，分別置放1、8 以及16小時。而後，以螢光光譜儀於波長300~400nm偵測各該待測樣品之螢光強度，結果如第四圖所示，其中，以未添加任何藥物之樣品作為對照組。As in the procedure described in Example 3, the drug Nucleozin at a concentration of 4 μM was added to the recombinant nucleocapsid protein solution to form a sample to be tested, and placed at 1, 25 ° C, respectively, 1 and 8 And 16 hours. Then, the fluorescence intensity of each sample to be tested is detected by a fluorescence spectrometer at a wavelength of 300 to 400 nm, and the results are shown in the fourth figure, wherein a sample without any drug is used as a control group.
由第四圖之結果顯示，當各該待測樣品放置時間越長，所測得之螢光強度亦隨時間增加而減少，顯示色胺酸螢光探針係分佈於核殼蛋白，並且藥物Nucleozin能夠誘導其結構隨著時間而改變以及使螢光強度降低。The results of the fourth graph show that the longer the time of each sample to be tested is, the measured fluorescence intensity decreases with time, indicating that the tryptophan fluorescent probe is distributed in the nucleocapsid protein, and the drug Nucleozin is able to induce its structure to change over time and to reduce fluorescence intensity.
實例五：以重組核殼蛋白檢測藥物Nucleozin(三)Example 5: Detection of Nucleozin by Recombinant Nucleocapsid Protein (III)
如同實例三所述流程，分別將濃度為2μM、4μM、20μM、40μM之藥物Nucleozin添加於重組核殼蛋白溶液中，形成待測樣品，並於25℃下，分別置放1、8以及16小時。而後，以螢光光譜儀於波長331nm偵測各該待測樣品之螢光強度，結果如第五圖所示，其中，橫軸為不同放置時間，縱軸係由下列公式所計算得到之數值：(未加入藥物之待測樣品之螢光強度-各待測樣品之螢光強度)/未加入藥物之待測樣品所得之螢光強度。As in the procedure described in Example 3, the drug Nucleozin at a concentration of 2 μM, 4 μM, 20 μM, and 40 μM was added to the recombinant nucleocapsid protein solution to form a sample to be tested, and placed at 25 ° C for 1, 8 and 16 hours, respectively. . Then, the fluorescence intensity of each sample to be tested is detected by a fluorescence spectrometer at a wavelength of 331 nm, and the result is as shown in the fifth figure, wherein the horizontal axis is a different placement time, and the vertical axis is a value calculated by the following formula: (Fluorescence intensity of the sample to be tested without the drug - the fluorescence intensity of each sample to be tested) / Fluorescence intensity of the sample to be tested without the drug added.
由第五圖可知於藥物Nucleozin與核殼蛋白比值為0.5以及1時，螢光強度係隨著時間增加而增加，而當藥物Nucleozin與核殼蛋白比值為5以及10時，即使時間增加，螢光強度係維持一定值。因此，顯示出核殼蛋白係具有多個藥物鍵結點。As can be seen from the fifth figure, when the ratio of Nucleozin to nucleocapsid protein is 0.5 and 1, the fluorescence intensity increases with time, and when the ratio of Nucleozin to nucleocapsid protein is 5 and 10, even if time increases, The light intensity is maintained at a certain value. Thus, it has been shown that the nucleocapsid protein family has multiple drug binding sites.
實例六：以重組SOIV核殼蛋白檢測Nucleozin藥物Example 6: Detection of Nucleozin Drugs by Recombinant SOIV Nucleocapsid Protein
於本實例，如同實例三，利用藥物Nucleozin測試豬源性A型流感病毒之重組核殼蛋白之螢光促滅反應。In this example, as in Example 3, the fluorescent-killing reaction of the recombinant nucleocapsid protein of porcine influenza A virus was tested using the drug Nucleozin.
首先，分別取濃度為2μM、4μM、20μM、40μM之藥物Nucleozin，添加於最終濃度為4μM豬源性A型流感病毒之重組核殼蛋白溶液，形成待測樣品，於25℃下，放置三小時，其中，緩衝液之酸鹼值為7.5，係包含有50mM三羥基甲烷-氯化氫以及150mM氯 化鈉。以螢光光譜儀波長為300~400nm偵測各該待測樣品之螢光強度，其中，以未添加任何藥物之樣品作為對照組，結果如第六圖所示。First, the drug Nucleozin at a concentration of 2 μM, 4 μM, 20 μM, and 40 μM was added to a recombinant nucleocapsid protein solution with a final concentration of 4 μM of porcine influenza A virus to form a sample to be tested, and placed at 25 ° C for three hours. , wherein the buffer has a pH of 7.5 and contains 50 mM of trishydroxymethane-hydrogen chloride and 150 mM of chlorine. Sodium. The fluorescence intensity of each sample to be tested was detected by a fluorescence spectrometer wavelength of 300 to 400 nm, wherein a sample without any drug was used as a control group, and the results are shown in the sixth figure.
由第六圖之結果顯示隨著添加藥物Nucleozin之濃度增加，所測得之螢光強度隨之減弱，亦即藥物添加濃度與螢光強度間呈現反比趨勢。The results from the sixth graph show that as the concentration of the added drug Nucleozin increases, the measured fluorescence intensity decreases, that is, the drug addition concentration and the fluorescence intensity show an inverse trend.
實例七：比對重組H1N1核殼蛋白及重組SOIV核殼蛋白Example 7: Alignment of recombinant H1N1 nucleocapsid protein and recombinant SOIV nucleocapsid protein
取實例三與實例六各該待測樣品所測得之螢光強度，並以下列公式計算出一數值：(將未加入藥物之待測樣品之螢光強度-加入不同濃度藥物之各待測樣品之螢光強度)/未加入藥物之待測樣品之螢光強度Take the fluorescence intensity measured by the sample to be tested in Example 3 and Example 6, and calculate a value by the following formula: (The fluorescence intensity of the sample to be tested without adding the drug - each of the different concentrations of the drug to be tested Fluorescence intensity of the sample) / Fluorescence intensity of the sample to be tested without the added drug
而將所計算出之數據與各該待測樣品之濃度製作成一長條圖，如第七圖所示。And the calculated data and the concentration of each sample to be tested are made into a long bar graph, as shown in the seventh figure.
由第七圖之結果顯示，不論核殼蛋白係來自於台灣種新型流感病毒或是豬源性A型流感病毒，對於藥物Nucleozin會產生螢光猝滅反應，其中，又以新型流感病毒之核殼蛋白產生較為顯著之螢光猝滅反應，且經由中和試驗結果可發現藥物Nucleozin對於新型流感病毒較豬流感有更佳的病毒抑制效力。The results of the seventh graph show that whether the nucleocapsid protein is derived from a new type of influenza virus or a porcine influenza A virus in Taiwan, a fluorescent quenching reaction is produced for the drug Nucleozin, and the core of the novel influenza virus is also used. The capsid protein produced a more pronounced fluorescing quenching reaction, and the drug Nucleozin showed better virus inhibitory efficacy against the swine flu than the swine flu by the results of the neutralization test.
實例八：篩選未知化合物Example 8: Screening for unknown compounds
取二待測化合物1017及1018，其中，1017之化學結構如下所示：
1018之化學結構如下所示：
將待測化合物1017及1018分別以不同濃度2μM、4μM、20μM、40μM分別添加於最終濃度為4μM之新型流感病毒之重組核殼蛋白溶液中，分別形成待測樣品，放置3小時後，以螢光光譜儀偵測各該待測樣品之螢光強度，並再以實例七中所述之公式計算出各該待測樣品之螢光強度之比值，而結果如第八圖所示。The test compounds 1017 and 1018 were separately added to the recombinant nucleocapsid protein solution of the novel influenza virus with a final concentration of 4 μM at different concentrations of 2 μM, 4 μM, 20 μM, and 40 μM, respectively, to form a sample to be tested, and after being placed for 3 hours, The optical spectrometer detects the fluorescence intensity of each sample to be tested, and calculates the ratio of the fluorescence intensity of each sample to be tested according to the formula described in Example 7, and the result is as shown in the eighth figure.
由第八圖顯示化合物1018能夠顯著減少核殼蛋白之螢光強度，並隨著添加濃度之增加而減弱螢光強度，而化合物1017則無論於何種濃度下，皆無法顯著改變螢光強度。因此，可推論出化合物1018具有作為治療流感藥物之能力。From the eighth figure, it is shown that the compound 1018 can significantly reduce the fluorescence intensity of the nucleocapsid protein, and the fluorescence intensity is weakened as the concentration of the addition increases, and the compound 1017 does not significantly change the fluorescence intensity regardless of the concentration. Therefore, it can be inferred that the compound 1018 has the ability to treat influenza drugs.
實例九：化合物1017、1018之中和試驗Example 9: Compound 1017, 1018 neutralization test
本實例係藉由盤式螢光分析驗證化合物1017以及1018是否能夠抑制被流感病毒所感染之細胞的細胞病變效應。This example demonstrates by means of disc fluorescence analysis whether compounds 1017 and 1018 are capable of inhibiting the cytopathic effect of cells infected with influenza virus.
首先，將96孔之組織培養盤中置入200μl之腎細胞(MDCK)懸浮液，其中，該腎細胞懸浮液係以含10%牛血清之液體培養基(DMEM)稀釋成1x105 cells/ml，於37℃、5%二氧化碳之環境下培養16~20小時。以磷酸鹽緩衝液清洗細胞，加入150μl不含血清之培養基，內含有病毒液以及最終濃度達0.0002%之胰蛋白酵素，其中，感染劑量(multiplicity of infectivity(MOI)為0.2每個細胞。而後，再分別加入含有不同濃度之待測化合物1017、1018之50μl液體培 養基，於37℃、5%二氧化碳之環境下培養48小時後，將細胞以甲醛固定以及去活化病毒後，以0.1%結晶紫染色15分鐘，以清水清洗後測定於波長570nm下之吸光值，計算化合物降低病毒引發細胞造成病變現象50%之IC50 ，結果如第九圖A及第九圖B所示，其中，第九圖A係為化合物1018之結果，第九圖B係為化合物1017之結果，VC係為病毒感染之控制組。First, a 96-well tissue culture dish was placed in a 200 μl kidney cell (MDCK) suspension, and the kidney cell suspension was diluted to 1×10 5 cells/ml in a liquid medium (DMEM) containing 10% bovine serum. Incubate for 16 to 20 hours at 37 ° C in a 5% carbon dioxide atmosphere. The cells were washed with phosphate buffer, and 150 μl of serum-free medium containing virus solution and a final concentration of 0.0002% trypsin, wherein the multiplicity of infectivity (MOI) was 0.2 per cell, was added. Then, 50 μl of the liquid medium containing different concentrations of the test compound 1017, 1018 was separately added, and after culturing for 48 hours at 37 ° C in a 5% carbon dioxide atmosphere, the cells were fixed with formaldehyde and deactivated, and stained with 0.1% crystal violet. After 15 minutes, the absorbance at a wavelength of 570 nm was measured after washing with water, and the IC 50 of the compound to reduce the pathogenic phenomenon caused by the virus-inducing cells was calculated. The results are shown in Fig. 9 and FIG. A is the result of compound 1018, ninth panel B is the result of compound 1017, and VC is the control group of viral infection.
由第九圖A結果顯示化合物1018的確具有抑制細胞病變效應之功效，而由第九圖B可知化合物1017係無抑制細胞病變效應之功效。From the results of Fig. 9A, it was revealed that the compound 1018 did have an effect of inhibiting the cytopathic effect, and the ninth panel B showed that the compound 1017 had no effect of inhibiting the cytopathic effect.
由實例八及實例九可相互驗證出化合物1018的確具有抗流感病毒之能力，並亦證實藉由核殼蛋白所產生之螢光猝滅反應確實可以篩選出具有抗流感病毒功效之藥物。It can be mutually verified from Example 8 and Example 9 that Compound 1018 does have the ability to resist influenza virus, and it has also been confirmed that the fluorescent quenching reaction by nucleocapsid protein can indeed screen for drugs having anti-influenza virus efficacy.
由上述各該實例之說明，可知本發明所揭之抗流感候選藥物之篩選套組及其篩選方法係藉由核殼蛋白與待測藥物之結合程度，改變核殼蛋白所產生之螢光強度，而可得知待測藥物是否與核殼蛋白結合，篩選出具有抗流感病毒之藥物。因此，本發明所提供之抗流感候選藥物之篩選套組及其篩選方法係具有以下優點：From the description of each of the above examples, it can be seen that the screening kit for the anti-influenza drug candidate disclosed in the present invention and the screening method thereof change the fluorescence intensity generated by the nucleocapsid protein by the degree of binding of the nucleocapsid protein to the drug to be tested. However, it can be known whether the drug to be tested binds to the nucleocapsid protein, and a drug having an anti-influenza virus is selected. Therefore, the screening kit and the screening method thereof for the anti-influenza drug candidate provided by the invention have the following advantages:
其一、操作簡單，僅需將適當濃度之候選藥物添加入篩選套組，即可快速得知是否具有抗流感之功效，而縮短篩選候選藥物之流程。First, the operation is simple, and only the appropriate concentration of the candidate drug is added to the screening kit, so that it can quickly know whether it has the anti-influenza effect, and shorten the process of screening the candidate drug.
其二、可將製備完成之核殼蛋白溶液置放於多孔盤中，藉由多孔盤可同時地篩選大量候選藥物，節省反覆進行篩選實驗之時間以及所需人力。Secondly, the prepared nucleocapsid protein solution can be placed in a porous disk, and a large number of candidate drugs can be simultaneously screened by the porous disk, which saves time for screening experiments and manpower required.
其三、可準確篩選出能與核殼蛋白結合之候選藥物，以提昇新藥開發之成功率以及降低投入成本。Third, it can accurately select candidate drugs that can bind to nucleocapsid protein to improve the success rate of new drug development and reduce input costs.
上述說明係針對本發明之可行實施例之具體說明，為該實施例並非用以限制本發明之專利範圍，凡未脫離本發明技術特徵所為之等效實施或是變更，均應包含於本案之專利範圍中。The above description is directed to the specific embodiments of the present invention, and is not intended to limit the scope of the invention, and the equivalents and modifications of the present invention should be included in the present invention. In the scope of patents.
第一圖係為質體pET21b之圖譜。The first image is a map of the plastid pET21b.
第二圖係為電泳圖，其中，右邊為經純化新型流感病毒台灣種之重組核殼蛋白之電泳圖，左邊係為豬源性A型流感病毒之重組核殼蛋白之電泳圖。The second figure is an electropherogram, in which the right side is the electrophoresis pattern of the recombinant nucleocapsid protein of the purified influenza virus Taiwan species, and the left side is the electrophoresis pattern of the recombinant nucleocapsid protein of the porcine type A influenza virus.
第三圖係為添加不同濃度之藥物Nucleozin與新型流感病毒台灣種之重組核殼蛋白所被測得螢光強度所作成之曲線圖。The third panel is a plot of the measured fluorescence intensity of different concentrations of the drug Nucleozin and the recombinant nucleocapsid protein of the new influenza virus.
第四圖係為添加藥物Nucleozin經過反應不同時間與新型流感病毒台灣種之重組核殼蛋白所被測得螢光強度所作成之曲線圖。The fourth graph is a graph of the fluorescence intensity measured by the addition of the drug Nucleozin to the recombinant nucleocapsid protein of the novel influenza virus Taiwan species at different times.
第五圖係為於添加不同濃度之藥物Nucleozin並分別反應不同時間時，新型流感病毒台灣種之重組核殼蛋白所測得螢光強度，經計算後，以得到相對螢光強度之數值與藥物Nucleozin之濃度與反應時間所作成之直條圖。The fifth picture shows the fluorescence intensity measured by the recombinant nucleocapsid protein of the new influenza virus Taiwan species when different concentrations of the drug Nucleozin are added and reacted at different times. After calculation, the relative fluorescence intensity value and the drug are obtained. A straight bar graph of the concentration of Nucleozin and the reaction time.
第六圖係為係為添加不同濃度之藥物Nucleozin與豬源性A型流感病毒之重組核殼蛋白所被測得螢光強度所作成之曲線圖。The sixth figure is a plot of the measured fluorescence intensity of the recombinant nucleocapsid protein of different concentrations of the drug Nucleozin and the porcine influenza A virus.
第七圖係為添加不同濃度之藥物Nucleozin，與新型流感病毒台灣種之重組核殼蛋白與豬源性A型流感病毒之重組核殼蛋白分別被測得螢光強度而作成之直條圖。The seventh figure is a bar graph of the fluorescence intensity measured by adding different concentrations of the drug Nucleozin to the recombinant nucleocapsid protein of the novel influenza virus Taiwan species and the recombinant nucleocapsid protein of the porcine influenza A virus.
第八圖係為添加不同濃度之化合物1017以及1018，與新型流感病毒台灣種之重組核殼蛋白被測得螢光強度所作成之直條圖。The eighth figure is a bar graph of the measured fluorescence intensity of the recombinant nucleocapsid protein of the new influenza virus Taiwan species with different concentrations of compounds 1017 and 1018.
第九圖A係為化合物1017中和試驗之結果圖。Figure IX is a graph showing the results of the neutralization test of Compound 1017.
第九圖B係為化合物1018中和試驗之結果圖。Figure IX is a graph showing the results of the neutralization test of Compound 1018.
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| TW100123296A TWI479153B (en) | 2011-07-01 | 2011-07-01 | Screening kit for screening anti - influenza drugs and its screening method |
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| TW (1) | TWI479153B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200904399A (en) * | 2007-03-13 | 2009-02-01 | Adamas Pharmaceuticals Inc | Compositions and kits for treating influenza |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200904399A (en) * | 2007-03-13 | 2009-02-01 | Adamas Pharmaceuticals Inc | Compositions and kits for treating influenza |
Non-Patent Citations (1)
| Title |
|---|
| Kao RY et al., Identification of influenza A nucleoprotein as an antiviral target. Nat Biotechnol. 2010 Jun;28(6):600-5.Epub 2010 May 30. 陳冠任等人，高速篩選系統的建立與新藥物開發，化學66:4 1-7 ，2008.12 * |
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| TW201303297A (en) | 2013-01-16 |
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